1. Field of the Invention
The present invention relates to a novel in vivo system that can be used to screen or test therapeutic agents for treating neurodegenerative illnesses. Specifically, the in vivo system of the present invention is established by using zebrafish embryos coupled with following the neurite outgrowth from green fluorescent protein (GFP)—labeled neurons during zebrafish development.
2. Description of the Related Art
The assessment of neurite-promoting activity of growth factors and neurotrophins in vitro is usually done by the laborious process of counting neuronal processes manually under a standard microscope or confocal microscopy. But more recently, automated methods have become available. Automated monitoring and quantification of neurite formation and outgrowth in multiple samples have greatly enhanced therapeutic investigations in neuroscience, particularly in drug screening and the drug discovery process in general (Simpson et al., 2001). However, these methods still offer relatively low throughput in terms of detection when compared with other higher-throughput screens, and they are limited to targeted screens or purely secondary screens.
In neuroscience research, many projects are focused on identifying drugs that affect the growth of neurites. The discovery and characterization of compounds and new chemical entities that promote or suppress neuritogenesis are of great importance in the search for therapies to treat neurodegenerative illnesses, such as Alzheimer's disease and Parkinson's disease, as well as trauma that results in neuropathy and nerve injury, including stroke and spinal cord injuries (Makarovsky et al., 2003).
Heparin-binding Neurite-promoting Factor (HBNF)
Heparin-binding neurotrophic factor or neurite-promoting factor (HBNF) was first co-purified with bovine acidic fibroblast growth factor from brain tissues (Rauvala, 1989). It is a secretory heparin-binding protein with highly basic and cysteine-rich amino acid residues. In mammals, HBNF shares 50% identity with midkine (MK) and they constitute a new family of heparin-binding proteins (Kovesdi et al., 1990). HBNF and MK are not only functionally related proteins with similar promotion of neurite extension in PC12 cells (Kretschmer et al., 1991; Bohlen et al., 1991), but also structurally related proteins. They have two conserved cysteine residues, a highly conserved hinge region as well as two clusters of basic residues for heparin binding (Winkler et al., 2003). In addition to neurite outgrowth promotion, HBNF also has a variety of biological activities, such as stimulating cell growth, acting as an angiogenesis factor, and containing oncogenic activity. Therefore, it is also known as pleiotrophin (PTN) (Li et al., 1990) and heparin-binding growth-associated molecule (HB-GAM) (Merenmies et al., 1990).
Initially, the function of HBNF/PTN was found to promote neurite outgrowth from different cultured neuronal cell types, including primary embryonic cortical neurons (Hampton et al., 1992; Raulo et al., 1992), PC12 cells (Kuo et al., 1990) and neuroblastoma cells (Li et al., 1993). PC12 cells are derived from a transplantable rat pheochromocytoma with an important feature of responding to nerve growth factor (NGF) to differentiate into neuron-like cells. Upon exposure to NGF, PC12 cells cease proliferation and extend neuritis (Greene and Tischler, 1976). Without NGF, HBNF could induce weak but significant neurite extension in PC12 cells, when these cells were transfected with HBNF cDNA (Chang et al., 2004).
HBNF Receptor, syndecan-3, is a haparan sulfate proteoglycan
HBNF/PTN was first isolated as a heparin-binding protein with high affinity, suggesting that heparin or heparin-type carbohydrates may play important roles in the biological function of HBNF/PTN (Rauvala 1989). Indeed, further studies demonstrate that a transmembrane heparan sulfate proteoglycan, N-syndecan (syndecan-3), acts as a receptor for HBNF/PTN (Raulo et al., 1994). Both the heparan sulfate side chains of N-syndecan and polyclonal anti-N-syndecan inhibit HBNF/PTN-induced neurite outgrowth in the cultured neurons. In addition to N-syndecan heparan sulphate, the low molecular weight heparin displays more potent inhibition of HBNF/PTN-induced neurite outgrowth in cultured neuronal cells (Kinnunen et al., 1996; Rauvala et al., 1997). As mentioned above, we modified the in vivo neurite outgrowth assay in zebrafish embryos to investigate the inhibitory effect of heparin or heparan sulfate on HBNF-induced neurite outgrowth by further injection of different dosage of heparin or heparan sulfate into zebrafish embryo at two- or four-cell stage. In agreement with previous report (Rauvala et al., 1997), heparin could inhibit HBNF-induced neurite outgrowth in zebrafish embryos (data not shown). Therefore, the modified assay system also can be used to compare the inhibitory or enhancing effect of HBNF/PTN-induced neurite outgrowth in vivo by heparin and its modified forms, N-syndecan-derived saccharides, and other glycosaminoglycans.
Compounds with Enhancing or Inhibitory Effect on NGF-induced Neurite Outgrowth in PC12 Cells
Scoparia dulcis L. (Scrophulariaceae) is a widespread tropical herbaceous medicinal plant, which has been used widely as a traditional folk medicine for its antipyretic and analgesic properties and for its use in treating bronchitis and gastric disorders in South America. Three new acetylated flavonoid glycosides, 5,6,4′-trihydroxyflavone 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1→6)-beta-D-glucopyranoside (1), apigenin 7-O-alpha-L-3-O-acetylrhamnopyranosyl-(1→6)-beta-D-glucopyranoside (2), and apigenin 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1→6)-beta-D-glucopyranoside (3), were isolated from Scoparia dulcis together with the known compound eugenyl beta-D-glucopyranoside (4). Compounds 2 and 3 showed an enhancing activity of nerve growth factor-mediated neurite outgrowth in PC12D cells (Li et al., 2004).
Indocarbazostatins C (3) and D (4), new inhibitors of NGF-induced neurite outgrowth were isolated from culture broth of a mutant strain, Streptomyces sp. MUV-6-83 (Feng et al., 2004). The structural elucidation of 3 and 4 revealed that these inhibitors were methyl ester analogs of the corresponding ethyl ester compounds, indocarbazostatin (1) and indocarbazostatin B (2), respectively.
Zebrafish
Zebrafish is a good model organism for the study of vertebrate development (Penberthy et al., 2002; Rubinstein, 2003). The embryos develop outside the mother and are optically transparent, allowing direct observation of their embryonic development that takes only 48 hours for completion at 28° C. In my lab, we have cloned several zebrafish tissue-specific promoters including pancreatic-, neuron-, and muscle-specific promoters. Their tissue specificies of gene expression were confirmed by expression of GFP in zebrafish embryos. Therefore, these tissue-specific promoters could be used to drive GFP or RFP expression in zebrafish embryos. In general, it is common to investigate the function of known or novel genes by gain-of-function and loss-of-function in zebrafish. To achieve gain-of-function, genes of interest are driven by tissue-specific promoters and injected into one-cell zebrafish embryos (Gong et al., 2001). Alternatively, the expression constructs under the control of either ubiquitous or tissue-specific promoter were co-injected with tissue-specific promoter/GFP construct. On the other hand, to achieve loss-of-function, genes of interest are knockdowned by injection of morpholino antisense-oligomnucleotides (MAO) or coinjection of MAO with tissue-specific promoter/GFP construct (Nasevicius and Ekker, 2000; Urtishak et al., 2003). The suitable transgenic GFP/RFP zebrafishes also can be used to inject MAO or expression constructs, respectively.
Recently, many transgenic zebrafish with fluorescent organs for development of relevant models of human disease have been established (Shentu et al., 2003; Her et al., 2003; Her, et al., 2004). For example, transgenic lines of zebrafish with fluorescent blood vessels have been developed, which simplifies the process by which blood vessels are visualized (Lawson and Weinstein, 2002). This transgenic line was designed by driving expression of green fluorescent protein (GFP) with the fli-1promoter. Embryos and larvae with fluorescent blood vessels can be used for angiogenesis assays. The VEGF-specific tyrosine kinase inhibitor SU-5416 (semaxanib; SUGEN Inc) and the more broadly active tyrosine kinase inhibitor, SU-6668 (SUGEN Inc) have been found to inhibit angiogenesis in transgenic fluorescent embryos (Parng et al., 2002).
Enhancement of Neurite Outgrowth in Zebrafish Embryo
So far, there is no suitable in vivo assay to assess the enhancement of neurite outgrowth by neurite-promoting factors. In our lab, we used zebrafish embryos to establish an in vivo system that can be used to study the enhancement of neurite outgrowth from GFP-labeled live neurons during zebrafish development (Chang et al., 2004). Zebrafish embryos could provide more additional factors than those in PC12 cells for extensive neurite outgrowth. In zebrafish, HuC gene has been shown to be a useful early marker for neurons and a 2.8 kb promoter region of this gene is sufficient to direct GFP expression in a neuron-specific pattern closely similar to endogenous HuC expression (Park et al., 2000). As shown in the following data, pHuC-GFP alone displayed GFP expression in trigerminal gaglion, axon as well as rohon beard (RB) or motor neurons without visible branched and long neurites. However, coinjection of pcDNA-HBNF-HA and pHuC-GFP resulted in significant enhancement of neurite outgrowth with extensive branched and long dendrites. For the first time, these data indicated that the expression of zebrafish HBNF could induce robust neurite outgrowth with wider and complicate branches from GFP-labeled neurons during zebrafish development.